This invention relates to laser capture microdissection (LCM) in which direct extraction of cellular material from a tissue sample occurs to a transfer surface. The disclosure herein relates to isolating selected tissue samples to a film matrix in a format where the isolated sample segments can be conveniently collected for subsequent analysis.
Laser Capture Microdissection (LCM) has been described in Science magazine (Nov. 8, 1996 and Nov. 21, 1997). Summarizing, LCM typically involves placing a transporting substrate having an activatable coating for adhering to identified cellular material on a tissue sample. For example, a large (several cm2) piece of transparent, thermally activatable adhesive film (e.g. EVA polymer) is placed in close apposition with the upper tissue surface of a standard (desiccated) histopathology section (5-15 um thick) mounted on a glass microscope slide.
Once the identified cellular material of the tissue sample for micro-dissection is selected, typically by examination under a microscope, the substrate is then activated, typically be being pulsed with laser beam. The light energy is absorbed by the plastic film which melts in a small region. The activatable coating then flows onto and around microscopic tissue components thereby causing the film on cooling to be firmly adherent to the identified cellular material of tissue sample. Other target areas on the same slide can similarly treated.
When the film is subsequently lifted off the slide, the selected tissue comes with the film in a series of spots leaving the other tissue behind on the slide. Unfortunately, just as the identified cellular material was dispersely located on the slide, the identified cellular material is dispersely distributed on the transporting substrate.
In the ordinary case, the selected series of spots is then removed from the film by placing the film in a suitable reagent (e.g., proteinase K which digests the structural proteins of the tissue) which frees the molecules of DNA or RNA to be subsequently analyzed (using, e.g., PCR and gel electrophoresis). In order to confer specific transfer of single targets to a given reagent solution (i.e., specific xe2x80x9cmolecular extraction buffersxe2x80x9d), the transferred tissue spots and underlying attached film can be manually excised with a scalpel or scissors, or punched out with a precision punch/die directly into a cuvette.
With the development of this technique, demand has arisen for refinement. Accordingly, and in the text that follows, we identify the requirements of LCM and thereafter propose solution to those requirements. It is to be understood that in setting forth the requirements for laser capture microdissection, we claim invention. It goes without saying that the recognition of the problem to be solved can constitute part of the invention along with the solution to the problem once it is recognized.
In LCM, there is potentially a great research need (and eventual clinical need) to transfer smaller and smaller spot sizes (e.g. 25 microns or less). This leads to transfer of selected single cells or even organelles in order to study molecular modifications of specific cells within a pathology or cytology specimen. This poses two technology problems:
1) the accurate targeting, and adhesion of the film to specific cells based on microscopic observation, and
2) keeping the specificity of transfer high by minimizing the adjacent area of film transferred into the molecular extraction buffers.
In LCM, the spots in the film are randomly located roughly in a position corresponding to their location on the sample. When smaller spot sizes of sample are extracted, a problem which arises is finding them on the removed piece of film or transfer surface. Precise location is required so that the extracted portions of the sample can be precisely cut out. With precision cutting, the target tissue is completely recovered with minimal contamination from surrounding areas of film with low density nonspecific tissue adhesion.
Restricting LCM to the disclosed technology, precise computer control of the storage position and storage of coordinates may be practical. However, this computer control may loose the reliability of visual observation of the transfer process and may be complex and expensive. Further, computer location could fail to be accurate in many cases such as when the tape is distorted when it is lifted off the slide.
Nonspecific transfer to the film of un-targeted tissue in the region surrounding the identified cellular material is another problem. This non-specific transfer becomes increasingly important as spot size of targeted tissue is reduced. Ideally, no tissue outside the targeted region illuminated by the laser should be attached to the film upon its removal from the slide. Attachment of any undesired tissue would cause sample contamination with the desired tissue. As smaller and smaller target spots are used, the amount of stray tissue which can be tolerated becomes proportionally smaller.
Presuming that an extremely small contact area with a sample can be achieved, a problem then arises as to how to handle the transfer surface. Specifically, the positioning of a small spot of activatable transfer surface on the tissue at the target site, picking the small activated transfer surface off the tissue, and placing the isolated targeted cells in a cuvette, or storing them in a specifically identifiable manner without contamination by un-targeted tissue elements.
Having set forth the requirements, we now turn attention to a solution.
A method and apparatus of gathering by LCM identified cellular material from random locations on a tissue sample to designated locations on a transporting substrate enables convenient further processing. A transporting substrate has an identified mapped location for receiving identified cellular material. At least a segment of a selectively activatable coating is placed on the side of the transporting substrate in apposition to the tissue sample at the mapped location. The transporting substrate and sample are relatively moved to place the selectively activated coating at the mapped location in apposition to identified cellular material of the tissue sample which is to be extracted. Thereafter, the selectively activating coating is activated and impressed or impressed and activated to form an adhesive region on the transporting substrate for adhering to the identified cellular material. Upon removal of the transporting substrate from the tissue sample, identified cellular material adheres to the transporting substrate at the mapped location.
In a first embodiment, individual small pieces or coatings of selectively activated material are placed on a transporting film. For example, a selectively activatable coating is placed at the center of discrete pieces of film and exposed toward the sample. Each different piece of film is separately activated at its coated center by the laser. Apposition to the tissue during or after laser action occurs by using a pressure plate.
In an alternate embodiment, a continuous strip including a transparent substrate is used to hold the film, such as a continuous reel of tape with equally spaced, centrally located pieces of selectively activatable coatings. Thereafter, the continuous strip of transparent substrate is incrementally advanced so that center of the activatable coating is in the center of the microscope field. Adjacent bar codes or other optical encoded identifiers could serve to identify the individual transferred samples.
Sample collection can include a pressure plate actuated to hold the transporting substrate in contact with the tissue in the center of the microscope field before or after laser heating. After laser heating and attachment to the selected material from the sample, the pressure plate is raised, and the transporting substrate with the local activated coating with adherent tissue separated from the tissue sample. This process is sequentially repeated so that the transporting substrate is again advanced, the next piece of transporting substrate (clean) is put into the center of the field, pressed onto the tissue surface, and laser activated. This repetition not only advances the transporting substrate but also applies forces to reproducibly lift the targeted (adherent) tissue off the specimen slide. After the transporting substrate is advanced to the next clean (unused) spot, but before the film is pressed onto the specimen surface, the microscope stage and transporting substrate will be translated (in x and y) so that the next tissue target is in the center of the microscope field.
Where the transporting substrate is utilized with selectively coated spots, the substrate can be locally removed from the remainder substrate tape by punching or cutting off or peeling off the local regions of activated coating. This will leave attached identified cellular material localized to small pieces of transporting substrate. These small pieces of transporting substrate can be placed into a specific chamber or vessel for further processing and molecular analysis.
In another embodiment, the individual pieces of selectively activatable coating are mounted at the ends of arrays of deflectable struts attached to and projecting from a central common support, such as a wheel or a comb like structure. Each strut with its selectively activatable coating at the end acts as the transporting substrate and is sequentially indexed through the center of the microscope field. The ends of the struts serve as pressure plates, contacting the tissue with the activatable coating at the ends of the struts. The selectively activatable coat can be actuated when the strut is deflected, e.g., by a solenoid, (one spoke at a time, similar to a daisy wheel printer). After activation, the force on the strut is removed, allowing the strut to lift off the slide with the identified cellular material adhered to it. Thereafter, the strut array indexes the next strut with its piece of film into place, the stage can be moved to the next tissue target and the next strut is placed in contact with the new target.
Another variant of this approach is to use a comb shaped array (i.e, target film spots are in a linear array attached to a linear carriage by parallel spokes perpendicular to the array axis rather than the radial spokes of a xe2x80x9cdaisy wheelxe2x80x9d). A minute spot of film is placed at the end of each comb finger. A small solenoid deflects the finger so its end, which acts as a pressure plate, contacts the tissue in the center of the field. After laser heating, the solenoid force is removed, the finger returns to its undeflected position (lifting off the slide the tissue adhered to the film), and the comb is linearly indexed so a different finger/piece of film is in the center of the field and a different tissue target can be moved in place. Marks on the base of the comb could identify the samples. This technique allows extremely small and precise sample extraction.
Where the adhesive film is bonded to a pressure plate in the form of a deflectable strut, the strut at the attached identified cellular material can be broken off, and this broken off portion deposited into a capsule for collection and further processing.
It should be understood that in all the above collection techniques, other means exist to detach the adhesive spot from the substrate (e.g, pull tabs and peeling; specific solvents for the bond between the substrate and the adhesive spot, etc). All these improvements provide a means of removing precisely spaced (linear or angular separation) target film spots of small size which can easily be precisely positioned on the center of the optical field at a target site of the specimen already translated to this position under microscopic observation. Since the samples can be provided with identifying marks on the adjacent substrate, they can be stored in a compact manner as an array of sequential transfers (a daisy wheel, comb, or strip of tape substrate) until individually separated or grouped according to cell type from among a series of transfers and removed from their substrates.
In yet another embodiment, a piece of continuous transporting substrate is provided with a continuous central and narrow selectively activatable coating. This piece of continuous film is put in contact with the tissue at the narrow selectively activatable coating by a narrow (in the direction of the film travel) pressure plate to define the (small) area of contact. This technique avoids the problem of aligning, in the direction of film travel, a discrete spot of adhesive with the center of the field. As long as the narrow adhesive part of the tape passes through the field, and the stationary pressure plate is lined up with the fieldxe2x80x94both modest requirementsxe2x80x94alignment will be satisfactory. This approach has the advantage that it enables a small contact area of adhesive film on the tissue without: a) complications due to handling and alignment between the film and the target area, b) requiring specialized developments in the film above and beyond its basic adhesive properties, and c) requiring development of a specialized tape cassette e.g. with a built in pressure plate etc.